A key mechanism to immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is recombination at the vls locus. Recently, a targeted-deletion mutant of vls was generated that demonstrated its absolute requirement for persistence in mice. Despite this advance, there remains a fundamental gap in our understanding of the mechanism behind vlsE antigenic variation. Our long-term goal is to identify and characterize the Borrelia protein(s) responsible for antigenic variation of vlsE, as well as the mammalian host factor(s) involved in activating vlsE recombination. The objective of this application is to identify cis-acting DNA elements required for vls switching, and to establish the importance of the structure and position of the vls locus for vlsE recombination. The central hypothesis is that the 51 bp inverted repeat and 17 bp direct repeats of the B. burgdorferi vls locus act as cis-acting DNA elements that are essential for vls switching. Furthermore, we hypothesize that the position of the vls locus adjacent to the telomere end is necessary for efficient vlsE recombination, and that antigenic switching requires a cis organization of the locus. The rationale for the proposed research is that, once the key cis-acting elements and important structural components are defined, they will provide critical clues to the protein(s) involved with vlsE recombination. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by the published B. burgdorferi genome sequence and cited work by other groups, this hypothesis will be tested by pursuing two specific aims: 1) Identify the cis-acting DNA elements required for vlsE recombination;and 2) Establish the importance of vls locus structure and positioning for vlsE recombination. Under the first aim, mutations of these DNA repeat regions within the vls locus will be generated and cloned into an E. coli plasmid vector. These vls mutant plasmids will then be transformed into a vls-deficient strain of B. burgdorferi in order to reconstitute the vls locus, and transformants will be used to infect both immunologically-competent and -deficient mice. Under the second aim, DNA constructs with increased distances between the vls locus and the telomere end will be assayed for the ability to carry out vlsE recombination. In addition, spirochetes harboring the vlsE gene and silent cassettes on two separate linear DNA molecules will be examined for vls switching after being passaged through a mouse host. The proposed work is innovative, because it involves replacement of genetically manipulated versions of the complete locus into the vls-knockout clone of B. burgdorferi. When applied, the results from the proposed studies are expected to allow the targeting of this system in order to significantly reduce the ability of this pathogen to establish a persistent infection in the mammalian host. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important area of Lyme disease research that has potential applicability to understanding immune evasion and pathogenesis by Borrelia burgdorferi. The proposed research has relevance to public health because the resulting discoveries have the potential to fundamentally advance the field of B. burgdorferi immune evasion, and may have broad implications for antigenic variation systems in other animal and human pathogens. Thus, the findings are ultimately expected to be applicable to the health of human beings.